1. Field of the Invention
This invention relates to methods for the large-scale enzymatic deamidation of foot proteins. The deamidation proteins have utility as emulsifying or foaming agents in many food systems.
2. Description of the Prior Art
The functional properties of foot proteins depend on their conformation in food systems. The relationship of protein structure to functionality is such that altering the chemistry of food proteins can improve functional properties such as solubility, viscosity, gelation, fat emulsification and foaming. The conversion of protein amide groups of carboxyl groups by deamidation improves solubility and other physical properties of protein under mildly acidic conditions.
Improving solubility, emulsifying or foaming properties of edible proteins enchance their use as functional ingredients in many food systems, including beverages, pourable and nonpourable dressings, whipped toppings, frozen desserts, confections, baked goods and meat.
An enzymatic aproach to protein deamidation offers serval advantages over a chemical approach, including the speed of reaction, the fact that the reactions take place under mild conditions such as neutral pH and room temperature and most importantly, they are highly specific. The mild conditions reduce energy costs and the high specificity increases processing efficiency and minimize the need for downstream processing.
Hamada et al., (J. Food Sci. 53:1132; 1988) used the peptidoglutaminase from B. circulans todeamidation soy peptides and proteins. Peptidoglutaminase (PGase) readily deamidation soypeptides but its activity towards the intaaaact protein was small. They suggest that limited deamidation was due to the large molecular size and/or unique conformation of soy protein. There is a need to provide for an efficient enzymatic deamidation process for food proteins of large molecular size and unique conformation at near physiological pH.
Enzyme immobilization provides effective utilization and useful saving of enzyme in food processing. However, there are several disadvantages using immobilization techniques, including material cost, binding efficiency and the selection of an appropriate support. These drawbacks have made the technique difficult to apply commercially. Several investigators have employed ultrafiltration (UF) in enzyme immobilization and enzyme recovery for recycling processes. In comparison to coinventional immobilization techniques, UF methods can be simpler, less expensive and a more efficient. Additionally, using UF may improve yield, production consistency and product quality.
UF is used in the dairy industry for concentrating protein components in milk, for cheese manufacturing and for preparation of whey protein concentrates. Additionally, it has been used experimentally for separation of lysozyme egg white; clarification of juice; and removal of bacteria, viruses, and other organisms in the production of sterile products (Modler et al., Food Technol. 42(10):114; 1987).
PGase produced by Bacillus circulans has recently been used in the deamidation of casein and whey protein hydrolysates (Gill et al., Irish J. Food Sci. Tech. 9:30; 1985) and soy peptides and proteins (Hamada et al., J. Food Sci. 53:671 1988). Enzymatic deamidation of food proteins improves solubility and other functional properties of proteins under mildly acidic conditions (Hamada and Marshall, J. Food Sci. 54:598 1989; Kato et al., J. Food Sci. 54:1345 1989).
In rendering large molecular weight protein substrates for PGase deamidation, reducing protein size is an inevitable step in the process. Hamada and Marshall (1988) reported a quantitative relationship between the extent of deamidation and the degree of protein hydrolysis combined with heat treatment. Proteolysis provides polypeptides with different molecular sizes and many times smaller than the large molecular weight (MW) PGase. Kikuchi and Sakaguchi (Agri. Biol. Chem. 37:827; 1973) reported that the MW of PGase I and II, estimated by gel filtration to be 200 kd for both in a non-dissociating salt and pH consitions.
UF reactors are well-mixed reactors. Blatt et al., (Anal. Biochem. 26:151; 1968) reported the possibility of using ultrafiltration membranes to separate higher molecular weight biologically active substances from lower molecular weight solutes. Because of the selective nature of the UF membrane this type of reactor is most useful for carrying out enzyme reactions using membranes that are impermeable to the enzyme, but permeable to substrates and products. UF immobilization of enzymes offer several distinct advantages relative to other immobilization methods. Immobilization is achieved without chemical alternation of the enzyme and can be accomplished quickly and easily (Chambers et al., Meth. Enzymol. 44:291; 1976).
Butterworth et al (Biotechol. Bioeng. 12:615; 1970) were the first to use UF concept to immobilize alpha-amylase in a continuous reactor to hydrolyze starch. Abbott et al. (Biotechnol. Bioeng. 18:1033; 1976) reported a successful use of cephalosporin acetylesterase in an UF reactor.
Desselie and Cheryan (J. Food Sci. 46:1035; 1981) developed a continuous method for the enzymatic proteolysis of proteins in an UF reactor. There are major disadvantages using separated or immobilized biocatalysts such as enzymes. Losses in enzyme activity of 10-90% have been reported (Cheetham, "Handbook of Enzyme Biotechnology," A Wiseman (Ed.) Ellis Horwood Limited Publishers, Chichester and New York, p. 54; 1986). Additionally, product recovery has traditionally been low because of concentration polarization and membrane fouling.